Heterogeneous inseminate system

ABSTRACT

A heterogeneous inseminate including a first amount of sperm of a first animal and a second amount of sperm of a second animal of the same species, the first amount of sperm and the second amount of sperm sex-selected sperm of the same sex, useful in the in-vivo or in-vitro fertilization of an egg of a female animal of the same species for the production of sex-selected embryos and sex-selected offspring.

This United States Non-Provisional Patent Application is a continuation-in-part of U.S. patent application Ser. No. 12/806,945, filed Aug. 24, 2010, which is a continuation of U.S. patent application Ser. No. 10/523,268, filed Jul. 7, 2005, which is the National Stage of International Patent Cooperation Treaty Patent Application No. PCT/US03/24460, filed Aug. 1, 2003, which claims the benefit of U.S. Provisional Patent Application No. 60/400,971, filed Aug. 1, 2002, each hereby incorporated by reference herein.

I. TECHNICAL FIELD

A heterogeneous inseminate including a first amount of sperm of a first animal and a second amount of sperm of a second animal of the same species, the first amount of sperm and the second amount of sperm sex-selected sperm of the same sex, useful in the in-vivo or in-vitro fertilization of an egg of a female animal of the same species for the production of sex-selected embryos and sex-selected offspring.

II. BACKGROUND

Pre-selection of sex has been accomplished in many species of livestock following the development of safe and reliable methods of separating sperm cells (also referred to as “sperm”) into enriched X chromosome bearing and Y chromosome bearing populations. See for example methods and apparatus disclosed by WO 00/06193; WO 02/043574; WO 01/85913; WO 99/33956; WO 01/40765; WO 98/34094; WO 99/42810; WO 02/043486.

A significant problem with sex-selected sperm cells may be that separation of sperm cells at rates sufficient to produce sex-selected insemination samples or sex-selected inseminates which are viable or sufficiently fertile for commercial application by conventional technology has necessitated increasing fluid stream pressure of flow cytometers or flow sort instruments to about 50 pounds per square inch. With respect to sperm cells of many species of mammals entrained in fluid streams having flow characteristics resulting from this application of pressure, the viability, motility, or other fertility characteristics may be altered.

Another significant problem with sex-selected sperm cell inseminates or sex-selected sperm cell insemination samples can be the vast difference in sperm cell fertility characteristics which can vary greatly between samples. As such, success of artificial insemination performed under substantially identical conditions can result in correspondingly different pregnancy rates or cannot achieve the threshold of fertility in the context of conventional procedures for successful fertilization of eggs whether in-vitro or in-vivo.

Additionally, cryopreservation and thawing of sex-selected sperm can result in substantially reduced motility, percent intact acrosomes and post thaw survival time, all of which can militate against successful use of cryopreserved-thawed sex-selected sperm for the in-vitro or in-vivo fertilization of eggs of the same species of animal.

Additionally, certain animals, such as dairy cows, are known to have reduced fertility as compared to heifers. An adverse side effect of acquiring certain genetic traits advantageous to milk production can be concurrent with acquisition of genetic traits which lower fertility of dairy cows. Moreover, as to the example of dairy cows, it may also be desirable to increase the proportion of female offspring by the use of sex-selected sperm. In order to successfully breed animals having reduced fertility, especially in the instance of utilizing sex-selected sperm to increase the number of female offspring, it would be useful to have an inseminate, and a method of producing such inseminate, having increased fertility in order to increase pregnancy rates.

Therefore, a need exists for a method of increasing fertility of sperm within a sex-selected inseminate to overcome reduced fertility in inseminates whether due to low numbers of sperm contained in the inseminate, low fertility of sperm relating to animal to animal variation, the process of sex-selection, the low fertility of the female animal, or the like.

Additionally, a need exists for an assay from which fertility of sex-selected sperm cells can be compared directly in-vivo (for example, in conjunction with artificial insemination procedures) and in-vitro (for example, in conjunction with IVF procedures).

The instant invention addresses the need for a sex-selected inseminate having increased fertility to overcome reduced fertility due to limited numbers of sperm, low fertility of sperm relating to animal to animal variation or the process of sex-selection, and the low fertility of the female animal.

III. DISCLOSURE OF THE INVENTION

Accordingly, a broad object of the broad object of the invention can be to provide a heterogeneous inseminate (also referred to as a “heterogeneous insemination sample”) in which a first amount of sperm and a second amount of sperm obtained from a corresponding first animal and a second animal are combined, the first amount of sperm or the second amount of sperm or both being sex-selected sperm, resulting in an increase in fertility of the first amount of sex-selected sperm or the second amount of sex-selected sperm such that the egg(s) of an animal can be fertilized whether in-vitro or in-vivo by one of the sperm of the first amount of sex-selected sperm or second amount of sex-selected sperm with increased success rates as compared to conventional inseminates (also referred to as a “conventional insemination sample”) containing sex-selected sperm of only one animal, thereby increasing the number of embryos produced per unit of sex-selected sperm which can reduce the cost of embryo production.

Another broad object of the invention can be to provide a cryopreserved heterogeneous inseminate in which a first amount of sperm and a second amount of sperm obtained from a corresponding first animal and a second animal are combined, the first amount of sperm or the second amount of sperm or both being sex-selected sperm, resulting in an increase in fertility of the first amount of sex-selected sperm or second amount of sex-selected sperm upon being thawed, which can correspondingly increase in fertility in the first amount of sex-selected sperm or the second amount of sex-selected sperm or both such that the egg(s) of an animal can be fertilized whether in-vitro or in-vivo by one of the sperm of the first or second amount of sex-selected sperm with increased success rates as compared to conventional cryopreserved inseminates containing sex-selected sperm of only one animal, thereby increasing the number of embryos produced per unit of sex-selected sperm and can reduce the cost of embryo production.

Another broad object of the invention can be to provide devices and methods of using such devices to control sperm cell fertility characteristics of sperm cells isolated from semen obtained from a male of species of mammal, such as motility, viability, fertilization rate, cleavage rate, blastocyst rate, or the like.

Providing controlled sperm cell fertility in accordance with the invention can be achieved with the sperm cells obtained from numerous and varied species of mammals, including without limitation, mammals selected from the group consisting of a bovine species of mammal, an equine species of mammal, an ovine species of mammal, a canine species of mammal, a feline species of mammal, a swine species of mammal, a marine species of mammal, a deer species of mammal, a primate species of mammal, a goat species of mammal, or a species of mammal listed by Wilson, D. E. and Reeder, D. M., Mammal Species of the World, Smithsonian Institution Press, (1993), hereby incorporated by reference herein.

With respect to certain embodiments of the invention, controlled sperm cell fertility characteristics comprises affirmative selection of fertility characteristics in advance of isolating sperm cells from the semen of the male of the species of mammal and application of the invention to alter sperm cell fertility characteristics to provide the fertility characteristics desired. With respect to other embodiments of the invention, sperm cell treatment conditions are selected within a broader range of sperm cell treatment conditions that can be used treat sperm cells of a particular species of mammal to obtain sperm cells having controlled fertility characteristics. Controlled fertility characteristics can comprise a desired proportion of motile sperm cells, intact acrosomes, viable sperm cells within a population of treated sperm cells; or can comprise a desired cleavage rate of oocytes or rate of blastocyst formation when treated sperm cells are utilized to fertilize oocytes in vitro; or can comprise a desired pregnancy rate or sex ratio of offspring when treated sperm cells are utilized for artificial insemination.

With respect to certain embodiments of the invention, a greater proportion of motile sperm cells, a greater proportion of viable sperm cells, a greater proportion of intact acrosomes, or a greater number of fertile sperm cells within a treated sperm cell population can be achieved compared to conventional treatment of the same of sperm cell population. Certain embodiments of the invention allow provision of sperm cells having controlled fertility characteristics which are not substantially different than, or are substantially comparable to, the fertility characteristics of sperm cells in fresh ejaculated semen. In other instances, application of certain embodiments of the invention can, if desired, result in sperm cells having controlled fertility characteristics which are substantially different than those of sperm cells of fresh ejaculated semen. In particular, certain embodiments of the invention can be used to provide bovine sperm cells having controlled fertility characteristics or can be used to provide equine sperm cells having controlled fertility characteristics, which if desired can be provided with fertility characteristics substantially comparable to the fertility characteristics of bovine sperm cells or equine sperm cells within freshly ejaculated bovine or equine semen.

Another broad object of the invention can be to provide sperm cell insemination samples having controlled sperm cell fertility characteristics, such sperm cell insemination samples, without limitation, can be configured for artificial insemination of a female of a species of mammal, in vitro fertilization of oocytes, or intracytoplasmic injection of sperm cells, or the like.

Another broad object of the invention can be to provide methods of sex selecting sperm cells that can provide affirmative control of sperm cell fertility characteristics such as motility, viability, fertilization rate, cleavage rate, blastocyst rate, or the like. One aspect of this broad object of the invention can be to provide flow cytometry or cell sorting devices or methods of flow cytometry or cell sorting which allows affirmative control of the fertility characteristics of sex-selected sperm cells.

Another object of the invention can be to provide sex-selected bovine sperm cell insemination samples having controlled sperm cell fertility characteristics configured for artificial insemination of a female of a bovine species of mammal containing between about 100,000 and about 3,000,000 sex-selected bovine sperm cells having controlled sperm cell fertility characteristics.

Another object of the invention can be to provide sex-selected equine sperm cell insemination samples having controlled sperm cell fertility characteristics configured for artificial insemination of a female of an equine species of mammal containing between about 5,000,000 and about 50,000,000 sex-selected bovine sperm cells having controlled sperm cell fertility characteristics.

Another significant object of the invention can be to provide devices or methods of maintaining controlled sperm cell fertility characteristics of sperm cells with respect to processing of sperm cells, storage of sperm cells, or use of sperm cells, including, but not limited to, insemination of female mammal(s) or fertilization of oocyte(s).

Another significant object of the invention can be to provide methods of artificially inseminating females of a species of mammal with sperm cell insemination samples having controlled sperm cell fertility characteristics. With respect to certain embodiments of the invention, methods of insemination with a low or reduced number of sperm cells having controlled fertility characteristics compared to the usual number or typical number of sperm cells used in such artificial insemination procedures whether or not such sperm cells are separated into enriched X chromosome bearing or Y chromosome bearing sperm cell populations.

Another broad object of the invention can be to provide a method of assessing comparative fertility of sperm cell populations. Certain embodiments of the invention provide a method of assessing comparative fertility of sperm cells from different males of a species of mammal when sperm cells from each male are exposed to substantially the same flow cytometric treatment. Other embodiments of the invention provide a method of assessing comparative fertility of sperm cells from the same male of a species of mammal which are exposed to different flow cytometric treatments. Certain embodiments of the invention provide methods of showing comparative fertility of sperm cells having controlled fertility characteristics.

Naturally, further significant objects of the invention are made clear in the proceeding description of the invention.

IV. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a sorter system according to a flow cytometer separation technique for the present invention.

FIG. 2 is a diagram of the entrained cells in the free fall area of a typical flow cytometer.

FIG. 3 is a plot which shows the level of resolution achieved between an X-chromosome bearing population and a Y-chromosome bearing population resulting from the separation of sperm in a first amount of sperm obtained from a first male animal using a flow cytometer in accordance with the method shown in FIG. 9.

FIG. 4 is a bivariate plot which shows separation of sperm in a first amount of sperm obtained from a first animal separated into an X-chromosome bearing population and a Y-chromosome bearing population using a flow cytometer in accordance with the method shown in FIG. 9.

FIG. 5 is a plot which shows the level of resolution achieved between an X-chromosome bearing population and a Y-chromosome bearing population resulting from the separation of sperm of a first amount of sperm obtained from a first male animal combined with a second amount of sperm obtained from a second male animal using flow cytometry in accordance with the method shown in FIG. 8.

FIG. 6 is a bivariate plot which shows the level of resolution achieved between an X-chromosome bearing population and a Y-chromosome bearing population resulting from the separation of sperm of a first amount of sperm obtained from a first animal combined with a second amount of sperm obtained from a second animal using flow cytometry in accordance with the method shown in FIG. 8.

FIG. 7 is an illustration which shows an enlarged portion the bivariate plot shown in FIG. 6.

FIG. 8 is a block diagram which shows a particular method of how to make and use an embodiment of the inventive heterogeneous inseminate.

FIG. 9 is a block diagram which shows a particular method of how to make and use an embodiment of the inventive heterogeneous inseminate.

V. MODE(S) FOR CARRYING OUT THE INVENTION

Now referring generally to FIGS. 1-9, embodiments of the invention can include a plurality of amounts of sperm (1) from corresponding plurality of male animals (2) of a species of mammal, as broadly defined above. Particular embodiments also include a plurality of amounts of sperm (1) obtained from a species of bird or fish. As to particular embodiments, each of the plurality of amounts of sperm (1) separately, or in combination as further described below, can then be entrained in a fluid stream (3) having flow characteristics. A fluid stream (3) within a conduit has flow characteristics influenced by the rheological properties of the fluid stream (3), the configuration or geometries of the conduit in which the fluid stream (3) flows, as well as external forces applied to the fluid stream (3) such as hydrostatic pressure, oscillatory vibrations, piezoelectric vibrations, oscillations in heat, or the like.

Importantly, these flow characteristics of the fluid stream (3) contribute to the amount of pressure required to move fluid within the conduit. As a non-limiting example, in flow cytometry, fluid moves within a relatively large cross sectional area and then within a relatively small cross sectional area past an analysis interface to a final collection point. This type of configuration or geometry along with rheological properties of the fluid stream can create localized forces such as compressive forces, sheer forces, or the like, which can have influence the physical integrity of particles such as sperm cells (6) entrained in the fluid stream (3).

With respect to those embodiment of the invention which include components of or steps involving flow cytometry or cell sorting as a means to analyze, separate based upon a sperm cell characteristic, sex select, or other wise process sperm cells (6), a conceptual non-limiting flow cytometer or cell sort instrument is shown in FIG. 1 and in FIG. 2.

A flow cytometer (4) or cell sort instrument includes all or a portion of the components shown in FIG. 1, including without limitation, a sperm cell source (5) which acts to establish or supply sperm (6) to analyze, separate, control fertility characteristics, or be otherwise treated.

Sperm (6) can be deposited within a nozzle (7) in a manner such that each of the sperm (6) can be surrounded by a sheath fluid (8). Any sheath fluid (8) compatible with the flow cytometer (4) or flow sort instrument and which provides an acceptable environment for sperm (6) during flow analysis or processing can be utilized with the invention, including without limitation, sheath fluids (8) which contain, individually or in various combinations, a phosphate buffered saline, a citrate solution (such as a 2.9% sodium citrate solution), a HEPES buffered solution, or the like.

The sheath fluid (8) can be delivered from a sheath fluid source (9) so that as the sperm cell source (5) supplies sperm (6), the sheath fluid (8) can be concurrently fed through the nozzle (7). In this manner, the sheath fluid (8) forms a sheath fluid environment for the cells. Since the various fluids are provided to the flow cytometer (4) at some pressure, they flow out of nozzle (7) at a nozzle orifice (10).

By providing an oscillator (11) which may be very precisely controlled through an oscillator control (12), pressure waves may be established within the nozzle (7) and transmitted to the sheath fluid (8) exiting the nozzle (7) at nozzle orifice (105). Since the oscillator (11) thus acts upon the sheath fluid (8), the stream (3) exiting the nozzle orifice (10) eventually and regularly forms droplets (14). Because each of the sperm (6) are surrounded by a sheath fluid environment, the drops (14) may contain within them individually isolated sperm (6).

Since the droplets (14) generally contain isolated sperm (6), the flow cytometer (4), or cell sorter instrument, can distinguish between and separate droplets (14) based upon one or more distinguishing sperm cell characteristic(s) of each sperm (6) contained within a droplet (14). This is accomplished through a sperm cell sensing system (15). The sperm cell sensing system (15) involves at least some type of a detector (16) responsive to differences in sperm cell characteristic(s) of the sperm (6) contained within each droplet (14).

One type of sperm cell sensing system (15) is discussed at length in U.S. Pat. No. 5,135,759 to Johnson, hereby incorporated by reference herein. As the Johnson patent explains for sperm (6), the cell sensing system (15) may cause an action depending upon the relative presence or relative absence of a particular dye which may be excited by some stimulant such as the beam of a laser (17). While each of a plurality of amounts of sperm (1) can be stained with a dye, the differing length of the X-chromosome and the Y-chromosome causes different levels of staining. Thus, by sensing the degree of dye present in each sperm (6) it is possible to discriminate between X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19) by their differing emission levels. Alternate optics, detection and sperm cell analysis systems are known which can also be used in accordance with the invention and it is intended that the description provided by the Johnson patent is for illustrative purposes so that the numerous and varied uses of the invention can be understood. See also, WO 01/85913, hereby incorporated by reference herein.

In order to achieve the separation and isolation of the appropriate cells in a flow cytometer or cell sort instrument separation technique, the signals received from the detector (16) are fed to a sorter discrimination system (20) which very rapidly makes the decision and can differentially charge each droplet (14) based upon whether the desired cell does or does not exist within that droplet (14). In this manner the sorter discrimination system (20) acts to permit the electrostatic deflection plates (21) to deflect droplets (14) based on whether or not they contain the a sperm (6) having certain sperm cell characteristics. As a result, the flow cytometer (4) or cell sorter instrument acts to separate the sperm (6) by causing them to land in one or more collection containers (22). Thus by sensing some property of the sperm (6), the flow cytometer (4) or cell sorter instrument can discriminate between sperm (6) based on a particular characteristic and place them in the appropriate collection container (22). In certain flow cytometers (4) or cell sorter instruments, the droplets (14) entraining X-chromosome bearing sperm (18) can be charged positively and thus deflect in one direction, while droplets (14) entraining Y-chromosome bearing sperm (19) can be charged negatively and thus deflect the other way, and the droplets (14) in a waste stream (13) (containing unsortable cells) remain uncharged and are delivered undeflected into a suction tube or the like.

Now referring primarily to FIG. 2, the process can be further understood in that the nozzle (7) emits a stream (4) which because of the oscillator (11) (not shown in FIG. 2) forms droplets (14). Since the cell source (5) (not shown in FIG. 2) may supply sperm (6) which as described by Johnson can be stained (or in certain embodiments of the invention unstained as when using differential interference contrast (“DIC”) technology), the light emission generated by the beam generated by laser (or illumination source when using DIC technology) incident upon the dye (the sperm head when DIC technology is utilized) is differentially received by detector (16) so that the existence or nonexistence of a charge on each droplet (14) as it separates from stream (3) can be controlled by the flow cytometer (4). This control results in positively charged, negatively charged, and uncharged droplets (14) based upon their content. As shown in FIG. 2, certain droplets (14) are shown as deflected drops (23). These deflected drops (23) are those containing sperm (6) which can be one or the other sex. They are then deposited in the appropriate collector (22) thereby generating a population of sex-selected sperm cells (24).

Whether the fluid stream (3) occurs within the context of a flow cytometer (4), cell sorter, or other device which entrains sperm (6) within a fluid stream (3), the flow characteristics of the fluid stream (3) can be characterized and adjustment means for altering flow characteristics of the fluid stream (3) can be introduced to increase or decrease forces such as compressive forces, sheer forces, or the like, such that particles, cells, or sperm cells entrained in the fluid stream (3) can be physically, physiologically, functionally, or mechanically altered.

As such a selectably adjustable range of fluid stream characteristics for a flow path can be generated using the adjustment means and can be expressed as an incremental measure. For example, alteration of fluid stream characteristics within a flow cytometer (4) or cell sort instrument context can be incrementally adjusted and measured in pounds per square inch (“psi”) and typically allow the incremental increase or decrease in the fluid stream pressure between about 20 pounds per square inch and 100 pounds per square inch with a commensurate increase or decrease in fluid stream or sheath fluid velocity.

In accordance with certain embodiments of the invention, sperm (6) of a particular species of mammal can be entrained in a fluid stream (3) having adjustable fluid stream flow characteristics. Fluid stream flow characteristics can then be selectably adjusted over an incrementally measured range in which the entrained sperm cells (6) remain viable. Sperm cell fertility characteristics are then assessed for each of a plurality of amounts of sperm (1) in correspondence to each of a plurality of flow characteristics generated within the measured range.

Subsequently, sperm cell fertility characteristics with respect to sperm (1) from a species of mammal or individual members of a species of mammal can be controlled and sperm cell insemination samples can be generated having the desired sperm cell fertility characteristics as further described below.

For example, a plurality of amounts of sperm cells (1) were obtained from each of six bulls. Each of the plurality of sperm cells (1) were stained with 125 μM Hoechst 33342 for 45 min at 34° C. The processed sperm cells (6) were bulk-sorted (passed through a flow cytometer (4) or cell sorter instrument without sorting into subpopulations), or sorted with a flow cytometer (4) having a nozzle (7) with an orifice with an internal diameter of 70 μM, into isolated populations of X-chromosome bearing sperm (18) or Y-chromosome bearing sperm (19) (or both) at about 95% accuracy with the fluid stream (3) having a pressure of at 30 psi, 40 psi, or 50 psi. Lowering the fluid stream pressure from 50 psi to 30 psi reduced sorting rate by only 2 to 3%.

The sperm cells (6) were subsequently cooled to 5° C. and concentrated by centrifugation, loaded into 0.25 ml artificial insemination straws (25) with about 2×10⁶ total sperm cells (6) per 100 μL column, and frozen using a vapor freezing method along with unsorted controls. The sperm cells (6) in the artificial insemination straws (25) were subsequently thawed.

Sperm cells (6) were then evaluated with respect to various sperm cell fertility characteristics blindly by two observers at 30 and 120 min post-thaw for progressive motility, as well as by flow cytometry 105 min post-thaw, for percent live sperm cells by PI stain, and by CASA analysis 120 min post-thaw using the Hamilton Thorne system. The entire procedure was twice replicated. Factorial ANOVA indicated that both bull and pressure effects were significant (P<0.005, Table 1).

TABLE 1 Responses of sperm post-thaw to different system pressures during sorting.^(a) Pressure (psi) Response 50 40 30 Unsorted Control 30 min motility (%) 44.7 48.6 49.6 52.1 120 min motility (%) 34.5 40.8 42.7 40.8 Live sperm (%) 51.7 55.7 57.8 58.5 CASA total motility (%) 25.1 37.2 40.9 34.8 CASAALH* 6.0 7.6 7.8 8.8 ^(a)All statistically significant, P < .005. *Amplitude of lateral head displacement.

Higher numbers mean less stiff and more normal motility. There were typical differences among bulls in all responses. However, the bull by treatment interactions were small with one exception, meaning findings apply similarly to most bulls in the population. The flow characteristics of the fluid stream (3) adjusted incrementally to increase pressure affected substantially all sperm cell fertility characteristics measured, however, only highly statistically significant ones are listed in Table 1.

As can be understood, there was significant change in sperm cell fertility characteristics between sperm cell samples taken at about 50 psi and at about 40 psi, and then a much smaller change between about 40 psi and about 30 psi, indicating that the effect on sperm cell fertility characteristics can not be assumed linear. For bovine sperm cells exposed to the flow characteristics described at 30 psi, sperm cell fertility characteristics were substantially the same as non-sorted controls or comparable to non-sorted controls, and for a few responses better, if sperm cells are to be used for insemination or artificial insemination of females of the bovine species. Similar procedures were conducted with sperm cells obtained from stallions with similar results and conclusions.

Sperm cell fertility characteristics can be controlled with respect to sperm cells obtained from mammals. For most species of mammals altering fluid stream characteristics to incrementally reduce fluid stream pressure, whether in the context of flow cytometry or otherwise, can result in a graded series of corresponding sperm cell samples having altered sperm cell fertility characteristics which may be used for a variety of procedures including artificial insemination, in vitro fertilization, or intracytoplasmic injection as described below.

The invention provides alternate tests to assess binomial responses such as pregnant/not pregnant, which typically require large numbers of animals per treatment to obtain statistical significance unless treatment differences are fairly large. One embodiment of the invention which can amplify differences in sperm cell fertility characteristics of sex-selected sperm due to treatment differences comprises competitive, or heterospermic, fertilization, mixing sperm of different treatments or males (whether or not sex-selected) (also referred to as a heterogeneous inseminate (26)) before insemination, and determining the proportion of embryos, fetuses or offspring derived from each male or treatment

For example, fertility after sex selection of sperm cells (6) by flow cytometry or by cell sorting for DNA content at two different fluid stream pressures can be assessed using heterospermic insemination using sex as the genetic marker. Sperm cells (1) from each of two bulls was sorted into populations of X-chromosome bearing sperm (18) or Y-chromosome bearing sperm (19), or both, at about 95% accuracy with the fluid stream (3) pressure adjusted to either 30 psi or 50 psi. After concentrating sex-selected sperm cells (24) post-sort by centrifugation, 1×10⁶ X-chromosome bearing sperm (18) sorted at 30 psi were placed in 0.25 mL straws with 1×10⁶ Y chromosome bearing sperm (19) sorted at 50 psi for each bull, as well as the converse in other straws: 1×10⁶ Y-chromosome bearing sperm at 30 psi plus 1×10⁶ X-chromosome bearing sperm at 50 psi. These sex-selected sperm cells (18)(19), along with unsorted controls, were then frozen, thawed some months later, and inseminated into the body of the uterus of 85 Holstein heifers either 12 or 24 h after observed estrus with subgroups balanced across two inseminators.

Two months post-insemination, 81% of the 43 heifers becoming pregnant had fetuses of the sex (determined by ultrasound) corresponding to the sex of sperm processed at 30 psi. This differed from the 50:50 sex ratio expected (P<0.01), if there was no difference in sperm cell fertility characteristics of sperm cells sorted at the two pressures. The pregnancy rate with sex-selected sperm cells (24) at 2×10⁶ sperm per dose was 51% (43/85); this was similar to the controls of 20×10⁶ unsexed sperm per dose from the same ejaculates, 39% (9/23).

Another embodiment of the invention provides a method altering the cleavage rate and rate of blastocyst formation using sperm sex-selected sperm cells (24) having controlled sperm cell fertility characteristics. Two bovine sperm cell samples each having controlled fertility characteristics were generated by flow sorting bovine sperm cells at 40 psi and 50 psi respectively. Dose response of sperm cell concentration in the fertilization medium was conducted with X-chromosome bearing sperm cells (18) and Y-chromosome bearing sperm cells (19) from each sperm cell sample. Thus, a multifactorial procedure comprising 2 fluid stream pressures, 3 sperm cell concentrations (1, 0.33 and 0.11×10⁶ sperm/mL), 6 bulls and 2 sexes can be conducted.

About 2,000 oocytes (27) (also referred to as “eggs”) were aspirated from about 2 mm to about 8 mm follicles from slaughterhouse ovaries. Chemically defined media (CDM) were used throughout as described by Journal of Animal Sciences, 78:152-157 (2000), hereby incorporated by reference herein. Maturation took place in M-CDM supplemented with 0.5% FAF-BSA, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 0.1 μg/mL E₂, 50 ng/μL EGF and 0.1 mM cysteamine for 23 h at 38.8° C. and 5% CO₂ in air. Sex-selected sperm cells (24) frozen with 2×10⁶ cells per artificial insemination straw (25) were thawed and centrifuged at 400 g through 2 mL 45% and 2 mL 90% Percoll gradients for 20 min. Then the supernatant was discarded and 2 mL of FCDM supplemented with 0.5% FAF-BSA, 2 mM caffeine and 0.02% heparin was added to the sperm pellet and centrifuged at 500 g for 5 min. The supernatant was discarded leaving approximately 50 μL of sperm suspension. Matured oocytes (27) were washed once in FCDM and transferred in groups of 15 in 5 μL into 25 μL drops of FCDM under mineral oil. Fertilization took place by adding 10 μL of sperm suspension per drop for 18 h at 38.8° C., 5% CO₂ in air. Presumptive zygotes were cultured in CDM1 for 2 d and CDM2 for 4.5 d at 38.5° C., 5% O₂, 5% CO₂ and 90% N₂. On day 7.5, blastocyst development was evaluated: Quality 1 to 4 (1 being excellent and 4 being poor) and stage of development, 6 to 8 (6 full, 6.5 expanding, 7 expanded, 7.5 hatching and 8 hatched blastocysts). Data (Table 1) were analyzed by ANOVA and first deviation after arc sin transformation.

Cleavage (53.6 and 43.6%) and blastocyst (18.2 and 14.7%) rates were higher for procedures utilizing sperm cells having controlled sperm cell characteristics obtained at about 40 psi than at about 50 psi (P<0.01). There was no interaction between dose and pressure; therefore, there was a similar advantage to lower pressure at each sperm concentration. A clear dose response of sperm cell concentration for cleavage and blastocyst production was found (Table 2). Also, there were large differences among bulls (P<0.01) for both responses, and there was a bull×dose interaction (P<0.01) for % cleaved. The data indicate that the sperm dose should be >1×10⁶/mL for some bulls. Embryo quality was higher (P<0.01) for Y-chromosome bearing sperm (19) than for X-chromosome bearing sperm (18) (1.12 vs 1.57). Others have noted this for in-vitro fertilized embryos (28) when embryos were sexed, and this effect now is confirmed with sex-selected sperm (24).

TABLE 2 Cleavage (%, C) and blastocysts (%, B) per oocyte data presented by bull. Bull Sperm H023 H024 H025 H026 H027 H028 Avg conc. (10⁶) C B C B C B C B C B C B C B 0.11 18 1 6 1 4 11 36 15 20 7 54 15 30^(a)  8^(a) 0.33 44 4 7 2 72 31 62 21 29 11 68 18 47^(b) 14^(b) 1.0 56 18 35 14 85 43 83 34 72 27 85 29 69^(c) 28^(c) ^(a,b,c)Values without common superscripts within groups differ, P < 0.01.

Fertility of sex-selected sperm (24) can be been low compared to unsorted control sperm, due partly to mechanical damage during sperm sorting by flow cytometry, as above-described. Lowering system pressure improved both sex-selected sperm cell (24) quality and fertility in IVF. The present study evaluated the effect of system pressure during sperm sorting and extended maturation of oocytes (27) on development of sex-selected embryos (8) after ICSI. Sperm from each of 3 bulls were stained with 125 μM Hoechst 33342 for 45 min at 34° C., sorted into populations of X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19) at 95% accuracy with the pressure of SX MoFlo®sorters at 40 or 50 psi, and then cryopreserved. Fifty bovine oocytes (27) obtained from slaughterhouse ovaries were placed per well with 1 mL of CDM1 supplemented with 0.5% FAF-BSA, 2 mM glucose, 50 ng/mL EGF, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 1 μg/ml E2 and 0.1 mM cysteamine, and then matured for 24 h or 30 h at 38.5° C., 5% CO₂ in air. Cumulus cells of matured oocytes (27) were removed by vortexing, and oocytes (27) with a polar body were selected. Motile sex-selected sperm cells (24) from sorted frozen-thawed semen were recovered by centrifugation through 2 mL each of 45 and 90% Percoll, and the concentration adjusted to 4×10⁶/mL. Matured oocytes were divided into two injection groups, ICSI and sham injection using a Piezo injection system. The outer diameter of the sperm injection pipette was 8-10 μM. All manipulations were performed at room temperature (24-25° C.). After injection, oocytes (27) were activated with 5 μM ionomycin for 4 min, cultured in 50 μL of CDM1 at 38.5° C. under 5% CO₂, 5% O₂ and 90% N₂, and assessed for cleavage at 72 h post-injection. Uncleaved oocytes from ICSI and sham injected groups were stained with orcein and evaluated for fertilization status. Cleaved embryos were further cultured and blastocyst development was evaluated on day 7.5 after injection. Data were subjected to ANOVA; the arc sin transformation was used for percentage data.

With 24 h matured oocytes (27), there were no differences (P>0.1) between sex-selected sperm (24) sorted at 40 versus 50 psi for cleavage or blastocyst rates, nor was there pressure×bull interaction. There were significant effects of bulls for all responses studied (P<0.05). When injected with sex-selected sperm (24) sorted at 40 psi, oocytes (27) matured for 30 h resulted in a higher cleavage rate than 24 h matured oocytes (22.9 versus 12.2%, P<0.05), with no difference P>0.1) in blastocyst rate. Overall blastocyst development was higher in ICSI than in sham injection (7.5 versus 1.3%, P<0.05). When uncleaved oocytes from 24 h maturation were evaluated for fertilization status, ICSI showed higher percentage with 2 polar bodies and/or decondensed sperm compared to sham injection (15.7 versus 1.7%, P<0.05). With 30 h matured oocytes there was no difference in fertilization status between those two groups. We conclude that there was no difference in cleavage or development to blastocysts after ICSI using motile sex-selected sperm (24) that had been sorted at 40 psi vs 50 psi.

In another embodiment of the invention, heterospermic insemination with heterogeneous inseminate (26) using sex as the genetic marker can be used to rank fertility of males and to rank fertility of sperm treatments not involving sperm sexing. Current in-vitro tests of sperm function are not highly correlated with male fertility, and homospermic inseminations require hundreds of inseminations per treatment to obtain accurate fertility data. Heterospermic insemination with heterogeneous inseminates (26) generated by mixing the sperm of two or more males, provides an accurate estimation of relative fertility in most species examined.

Frozen, flow-sorted sperm from 4 groups of 4 bulls (2) were thawed and inseminated into heifers 12 h or 24 h following onset of estrus in all combinations of 3 bulls within groups (ABC, ABD, ACD, BCD). Equal numbers of progressively motile sex-selected sperm (24) were inseminated from each bull, totaling 600,000 motile sex-selected sperm (24) post-thaw. Half of each heterogeneous inseminate (26) was deposited into each uterine horn. Embryos were collected nonsurgically 14.5 to 20 days following estrus. Collections yielded 165 elongating embryos from 332 heifers (48%). Polymorphic DNA markers were used to genotype embryos to determine the sire of each embryo biopsy. After genyotyping, 118 of the 165 embryos could be assigned a specific sire. Heterospermic indices for ranking each bull group were calculated using the maximum likelihood analysis theorem. Each bull within groups was ranked based on these indices (Table 3). In group 1, the fertility of the poorest bull was significantly lower (P<0.05) than two other bulls. In group 2, the dominant bull had the highest index (P<0.05). Similar distinctions could be made in groups 3 and 4. However, in three of the groups the fertility of some bulls was not clearly high or low (P>0.05).

TABLE 3 Heterospermic indices ± SE for individual bulls within groups. Group 1 Group 2 Group 3 Group 4 1.47 ± .41^(a) 2.43 ± .43^(a) 1.68 ± .44^(a  )   0.92 ± .36^(a,b)   0.44 ± .27^(a,b) 0.22 ± .15^(b) 1.09 ± .39^(a,b) 0.46 ± .20^(a) 1.84 ± .46^(a) 0.90 ± .35^(b) 0.83 ± .31^(a,b) 2.02 ± .40^(b) 0.25 ± .17^(b) 0.45 ± .23^(b) 0.40 ± .22^(b  ) 0.59 ± .24^(a) ^(a,b)Indices without common superscripts differ, P < 0.05.

With these procedures, an average of 30 genotyped in-vivo embryos (8) per group of 4 bulls enabled detection of bulls with clearly differing fertility. Sperm treatments also could be evaluated with this technique. This in-vivo test requiring few female animals (29) rapidly provides information concerning which bulls have relatively high or low fertility.

The population of calves (30) (also referred to as “offspring”) obtained by artificial insemination (31) of female animals (29) with sex-selected sperm cells (24) in accordance with the invention are virtually identical to controls using unsex-selected sperm cells. Furthermore, artificial insemination (31) of female animals (29) with sex-selected sperm (24) resulted in approximately 90% of calves (30) of the planned sex. As described above, sperm cells (6) can be sex selected on the basis of DNA content by flow cytometry or by cell sorting after staining with H33342. The sex-selected sperm cells (24) can then be cryopreserved as described in Theriogenology, 52:1375 (1999), hereby incorporated by reference herein.

Estrus can be synchronized in heifers and cows (female animals (29)) of various beef and dairy breeds, either by feeding 0.5 mg melengestrol acetate (MGA) daily for 14 d followed by 25 mg prostaglandin F₂ (PGF₂) im 17 to 19 d later or injection of 25 mg PGF₂ im at 12-d intervals. Artificial insemination (31) with either frozen-thawed sex-selected insemination samples or frozen-thawed sperm from the same ejaculate have been accomplished at either 12 h or 24 h after initial observation of estrus. For each breeding group, about ⅔ of the inseminations were with sexed sperm while control sperm were used in the remainder. Pregnancy and fetal sex were diagnosed by ultrasound 2 months later.

Cattle were managed at 13 farms through calving and weaning under differing levels of management (N=49 to 228 per farm). Data collected included gestation length, birth weight, calving ease (1=normal to 4=Caesarian), weaning weight, neonatal deaths, and deaths from birth to weaning. Not all farms recorded birth and weaning weights. Data were subjected to factorial analysis of variance with factors: management groups, sex-selected sperm cells (24) versus control sperm, and sex of calves (30). The arc sin transformation was used for percentage data. Least-square means are in Table 4.

TABLE 4 Calving results from sexed and control calves. Gestation Neonatal Calving Birth Live at Weaning Treatment N length (d) death (%) ease weight (kg) weaning (%) weight (kg) Sexed^(a) 574 279 3.9 1.31 34.3 92.0 239 Control 385 279 5.9 1.30 34.1 88.9 239 ^(a)No significant differences (P > 0.1) for any response.

There were no differences (P>0.1) between calves from sexed versus control groups for any response studied, nor were there significant interactions. There were significant effects of management groups for all responses studied (P<0.001 for all except % alive at weaning, P<0.02). Also, there were significant differences (all P<0.001) between female and male calves (30) for birth weight: 32.2 and 35.5 kg; weaning weight: 232 and 246 kg; calving difficulty: 1.20 and 1.42; and gestation length: 278 and 280 d. The sex ratio of the control calves (30) was 51.0% males (N=382). Sorted populations of X-chromosome bearing sperm (18) resulted in 87.7% females, while sorted populations of Y-chromosome bearing sperm (19) produced 93.6% males (N=94). A few calves that were dead at birth did not have sex recorded and are not included. The recent development of flow cytometric separation of stallion spermatozoa has resulted in the production of normal foals with preselected sex. (Lindsey A. C., Morris L. H., Allen W. R., Schenk J. L., Squires E. L., Bruemmer J. E. Equine vet J. 2002, 34: 128-132).

For this technology to be accessible, semen will be transported from the flow cytometer (4) to the mare. This study examined the longevity and acrosome status of fresh stallion spermatozoa after sex pre-selection. Three ejaculates from each of 7 stallions were collected by artificial vagina and shipped to the laboratory at 20° C. for 2-6 h in a skim milk-glucose extender (1:1 v/v). The semen was centrifuged at 400 g for 10 min and the seminal plasma removed. The sperm pellet was re-suspended to 100×10⁶/mL in Kenneys modified Tyrodes medium (KMT), stained with Hoechst 33342 (5 mg/mL, Sigma-Aldrich, St. Louis Mo.), incubated for 30 min and subjected to flow cytometry. The sex-selected sperm cells (24) were centrifuged and resuspended to 40×10⁶ mL in KMT in 250 μL aliquots for 48 h storage at either 4° C. or 20° C. The total progressive motility (TPM) and the acrosome status of the sex-selected sperm cells (24) were evaluated prior to sorting and at 0, 2, 12, 24, 36 and 48 h after sorting. The TPM was evaluated microscopically and acrosomes stained with FITC-PNA (Sigma-Aldrich) and classified as intact, patchy or lost. The effect of stallions, time and storage temperatures were analyzed using the Proc GLM procedure and least means comparisons made (SAS Institute).

There was an effect of stallion (p=0.03) on sperm motility and on the proportion of intact acrosomes over time. Staining and incubating the spermatozoa with Hoechst 33342 resulted in a decrease in the proportion of intact acrosomes (Table 5). However, the proportion of intact acrosomes observed after sorting was higher than in the sperm population prior to sorting. The proportion of intact acrosomes declined (p<0.0001) as the lost acrosomes increased (p<0.0001) during 48 h after sorting, but there was no effect of time on the proportion of patchy acrosomes. There was a significant effect of sperm storage temperature after sorting such that that storage for 12 h at 20° C. resulted in higher motility than storage at 4° C. Sex-sorting spermatozoa by flow cytometry results in the selection of a population of spermatozoa which can maintain acrosome integrity for 24 h, equivalent to fresh spermatozoa. The maintenance of sperm longevity for 12 h after FACS separation should enable sex-sorted spermatozoa to be shipped to mares located some distance from the site of the flow cytometer.

TABLE 5 The motility and acrosome status of flow sorted spermatozoa over time. Stage of Processing TPM Acrosome intact Prestain 50.7 ± 10.2 57.1 ± 28.2 Post stain 42.0 ± 17.1 42.3 ± 26.7 Post incubation 48.0 ± 15.7  34.9 ± 27.1^(a)  0 h Post sort 49.9 ± 18.3  60.2 ± 22.3^(b)  2 h  4° C. 34.3 ± 19.3 47.1 ± 28.3 20° C. 40.8 ± 21.8 53.9 ± 24.5 12 h  4° C.   8.5 ± 12.8^(a) 59.8 ± 19.7 20° C.  27.0 ± 21.0^(b) 66.6 ± 12.0 24 h  4° C.  4.8 ± 12.9 56.2 ± 16.8 20° C. 17.2 ± 17.9 64.3 ± 16.4 48 h  4° C. 0.0 ± 0.0 32.7 ± 25.0 20° C. 5.1 ± 8.6 41.6 ± 24.0 ^(a,b)Values within a column with different superscripts are significantly different (p < 0.05).

Foals of predetermined sex have been accurately and reliably produced in a research setting (Lindsey et al., Equine Vet. J. 2002, 34: 128-132). Sex-sorted sperm would be more efficiently utilized by the industry, however, if froze-thawed sex-sorted sperm were available. The objective of this study was to compare the motion characteristics of sperm that had been stored for 18 h at 15° C., flow-sorted, and then frozen, to sperm that had been cryopreserved immediately following shipment at 18 h at 15° C. Two ejaculates were used from each of five stallions. Following collection, sperm for both treatments were extended to 25×10⁶/mL in a Kenney+Modified Tyrodes (KMT) medium and stored in a water bath at 15° C. for 18 h. After storage, sperm were allowed to reach ambient temperature (about 22° C.) prior to centrifugation at 600 g for 10 min. Seminal plasma was removed and the sperm pellet re-suspended to 500×10⁶/mL in KMT. An aliquot of sperm was removed (Control) from this sample, extended to 87×10⁶/mL in a skin-mild, egg yolk freezing extended (4% glycerol; FR5), and allowed to slow cool to 5° C. for 90 min before freezing in liquid nitrogen vapor. A second aliquot (Flow-sorted) of sperm was extended to 100×10⁶/mL in KMT, stained with Hoechst 33342 (5 mg/mL, Sigma-Aldrich, St. Louis Mo.), incubated for 30 min, and subsequently sorted by flow-cytometry. Sorted sperm was centrifuged at 850 g for 20 min, resuspended to 87×10⁶/mL in FR5, and allowed to cool slowly to 5° C. for 90 min prior to cryopreservation. Sperm for both treatments were packaged in 0.25 mL straws, and each straw contained 20 million sperm. Sperm were evaluated (blindly) for visual progressive motility (2 observers) at 30 and 90 min post-thaw. An aliquot of sperm from each straw was diluted in both KMT and in KMT containing 2 mM caffeine. Samples were allowed to equilibrate for 5-10 min at 37° C. prior to evaluation. A second straw of each treatment was evaluated (with and without caffeine) using the Hamilton-Thom Motility Analyzer (CASA). Results are in Table 1. Differences in motion parameters were determined by ANOVA. According to most measured responses, flow-sorting was detrimental to sperm motility. Additionally, 2 mM caffeine improved many sperm responses. There was an interaction whereby caffeine improved some responses more for sorted sperm than for control sperm. Therefore, the damage caused by sorting can be partially compensated for by caffeine. It is possible that similar compensation may occur in the mare reproductive tract Studies are currently in progress to compare the fertility of stored, cryopreserved stallion sperm to that of sperm that has been stored and sorted prior to cryopreservation.

TABLE 6 Vis Vis CASA CASA Treatment 30 90 Tot Prog VAP VSL VCL ALH BCF STR LIN C-Control 50^(a) 41^(a) 64^(a) 60^(a) 94^(a) 80^(a) 164^(a) 6.19^(a) 33^(a) 83^(a) 50^(a) Control 45^(a) 40^(b) 50^(b) 44^(b) 82^(a) 69^(a) 144^(a) 5.73^(a) 33^(a) 82^(a) 50^(a) C-Sorted 32^(b) 31^(c) 32^(c) 21^(c) 48^(b) 38^(b)  98^(b) 4.75^(b) 41^(b) 73^(b) 39^(b) Sorted 18^(c) 16^(d) 24^(d) 12^(d) 39^(b) 30^(b)  80^(b) 4.51^(b) 37^(c) 69^(b) 38^(b) ^(a,b,c,d)Values in the same column without common superscripts differ (P, 0.05). C-treatments stimulated with 2 mM caffeine.

Cow elk 3-6-yr of age in Colorado and Minnesota were synchronized for estrus in September by insertion of a progesterone CIDR into the vagina for 12-14 d. Upon removal of the CIDR, 200 IU of eCG was administered intramuscular and elk were timed-inseminated 60 h later. Fresh semen was collected via electro-ejaculation from a 5-yr old bull elk and slowly cooled over 4 h to about 20° C. for transportation as a neat ejaculate to the sperm-sorting laboratory. The ejaculate was concentrated to 1×10⁹ sperm/mL for straining by centrifuging 1.5 mL aliquots for 10 sec at 15,000×g. Semen was incubated in 112 μM Hoechst 33342 at 200×10⁶ sperm/ml in a TALP medium for 45 min at 34° C., and then diluted to 100×10⁶/mL for sorting. Sperm were sorted on the basis of differing DNA content of X and Y chromosome-bearing sperm. X chromosome-bearing elk sperm contain 3.8% more DNA than Y chromosome-bearing sperm. Sperm were flow-sorted over a 4 h period using MoFlo®SX operating at 50 psi with a TRIS-based sheath fluid. The 351 and 364 bands of an argon laser, emitting 150 mW, excited Hoechst 33342 dye bound to DNA. Both X and Y chromosome-bearing sperm were collected (about 92% purity as verified by reanalyzing sonicated sperm aliquots for DNA) were collected at about 4,700 sperm/sec into tubes containing 2 mL of 20% egg yolk-TRIS extender. Sorted volumes of 15 ml were sequentially collected. Approximately 110×10⁶ sperm of each sex were sorted and cooled to 5° C. Cover 90 min. An equal volume of glycerol (12%) containing extender was added to the sorted volume at 5° C. Sorted sperm aliquots containing 30-ml were concentrated by centrifugation at 4° C. for 20 min at 850×g. Sperm pellets were pooled, adjusted to 21.7×10⁶ sperm/mL and loaded into 0.25 mL straws. Each straw, containing 5×10⁶ total sperm, was frozen in liquid nitrogen vapor. As a control, 5×10⁶ total sperm from the same ejaculate were frozen in 0.25 mL straws at the same time as the sexed sperm. After thawing for 30 sec at 37° C., 65% and 60% of sperm (control and sexed, respectively) were progressively motile as determined by visual estimates. Cows at 3 different locations and management schemes were inseminated using routine trans-cervical semen deposition in the uterine body. Pregnancy was determined 40 d post insemination by assaying blood for Pregnancy-Specific Protein B (Bio Tracking, Moscow, Id.). Ten cows at one location were in poor condition at the time of insemination and no pregnancies were achieved with sexed or control sperm. The pregnancy rate at the other locations with sexed sperm (61%; 11/18) was similar to that for control inseminates (50%; 3/6). These pregnancy rates (sexed and controls) resulted from fewer sperm than are used in normal elk artificial insemination. Nine of eleven (82%) of sexed calves were of the predicted sex.

Now referring primarily to FIGS. 3, 4, and 9 a univariate plot (or histogram) (32) (FIG. 3) and a bivariate plot (33) (FIG. 4) can be generated using a flow cytometer (4) to separate or sort a first amount of sperm (34) or a second amount of sperm (35) or each of a plurality of amounts of sperm (1) from a corresponding first male animal (36), second male animal (37) or plurality of male animals (2) into subpopulations of X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19) prior to being combined to achieve an embodiment of the sex-selected heterogeneous inseminate (26), as above described. The histogram (32) (FIG. 3) and the bivariate plot (33) (FIG. 4) show that the mixture of X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19) in each of a first amount of sperm (34) or second amount of sperm (35) or plurality of amounts of sperm (1) can be resolved into separate corresponding subpopulations of X-chromosome bearing sperm (18) and subpopulations of Y-chromosome bearing sperm (19).

Now referring primarily to FIGS. 5, 6, 7 and 8, a univariate plot (or histogram) (32) (FIG. 5) and bivariate plot (33)(FIG. 6) (see also FIG. 7 showing an enlarged portion of bivariate plot shown in FIG. 6) can be generated using a flow cytometer (4) to separate or sort X-chromosome bearing sperm (18) from Y-chromosome bearing sperm (19) in a heterogeneous inseminate (24) prior achieved by combination of a first amount of sperm (34) with a second amount of sperm (35) or by combination of a plurality of amounts of sperm (1) from a corresponding first male animal (36), second male animal (37) or plurality of male animals (2) of the same species, as above described (also refer to FIG. 8). Because of differences in the same sperm characteristics (38) between a first amount of sperm (34) obtained from a first male animal (36) and a second amount of sperm (35) obtained from a second male animal (37), or between a plurality of amounts of sperm (1) obtained from a corresponding plurality of animals (2) of the same species, the separation, selection, or sorting (depending upon the method) of the X-chromosome bearing sperm (18) from the Y-chromosome bearing sperm (19) in a heterogeneous inseminate (26) of unsorted sperm may not have been achieved prior to the instant invention.

Now referring to primarily to FIG. 8, particular embodiments of a sex-selected heterogeneous inseminate (39) and particular methods of preparing and using a sex-selected heterogeneous inseminate (39) are presented. A plurality of amounts of sperm (1) can be obtained from a corresponding plurality of male animals (2), each of the plurality of male animals (2) of the same species. The term “plurality of male animals” (2) can encompass two, three, four, five, six, or a greater number of male animals (2), as above described, for heterospermic insemination. The plurality of amounts of sperm cells (1) can be combined to achieve embodiments of the heterogeneous inseminate (26), as above described. The heterogeneous inseminate (26) can be divided to produce a plurality artificial insemination dosages (40) which can as to particular embodiments be contained one each in a corresponding number of artificial insemination straws (25), as above described. One or more of the artificial insemination doses (40) can be used for artificial insemination (31) of a female animal (29) of the same species for the production of in-vivo embryos (28) whether as single embryo pregnancies to generate offspring (30) for meat or animal replacement or multiple embryo pregnancies for multiple ovulation embryo transfer (“MOET”) by subsequent flushing of a plurality of in-vivo embryos (48) from the female animal (29). As to other embodiments, the heterogeneous inseminate (26) can be divided or used as a number of in-vitro fertilization doses (41) for in-vitro fertilization (42) of one or more eggs (27) obtained from a female animal (29) to produce a plurality of in-vitro fertilized embryos (28).

Again referring primarily to FIG. 8, while a plurality of amounts of sperm (1) from a corresponding plurality of male animals (2) is shown; a particular illustrative example useful in understanding the numerous and varied embodiments of the invention can include a first amount sperm (34) obtained from a first male animal (36) and a second amount of sperm (35) obtained from a second male animal (37). The first male animal (36) and the second male animal (37) being of the same species. The first amount of sperm (34) obtained from the first male animal (36) can be combined with the second amount of sperm (35) obtained from the second male animal (37) to produce a particular embodiment of the heterogeneous inseminate (26).

As to particular embodiments of the heterogeneous inseminate (26), the first amount of sperm (34) can be an amount of infertile sperm (43) while the second amount of sperm (35) can have an initial level of fertility (44) outside of the heterogeneous inseminate (26). Upon combination of the first amount of sperm (34) (infertile sperm (43) with the second amount of sperm (35), as concentration of the first amount of sperm (34) increases in the heterogeneous inseminate (26) as compared to the concentration of the second amount of sperm (35), the second amount of sperm (35) can exhibit by comparison to the initial level of fertility (44) an increased level of fertility (45) within the heterogeneous inseminate (26). Accordingly, the first amount sperm (34) can be sufficient in number or concentration within the heterogeneous inseminate (26) such that, the second amount of sperm (35) achieves by comparison to the initial level of fertility (44) an increased level of fertility (45).

As to other particular embodiments of the heterogeneous inseminate (26), the first amount of sperm (35) outside the heterogeneous inseminate (26) can have greater fertility than the initial level of fertility (44) of the second amount of sperm (35) outside of the heterogeneous inseminate (26). In combination, the second amount of sperm (35) can exhibit an increased level of fertility (45) within the heterogeneous inseminate (26). Accordingly, the heterogeneous inseminate (26) can afford a substantial advantage over conventional inseminates when the second amount of sperm (35) can confer desirable genetic traits, but has an initial level of fertility (44) outside of the heterogeneous inseminate (26) which may be unsuitable for use as an artificial insemination dosage (40) for the artificial insemination (31) of a female animal (29) or may be unsuitable for use as an in-vitro fertilization dose (41) for in-vitro fertilization (42) of eggs (27) obtained from a female animal (29). The comparably lesser initial level of fertility (44) of the second amount of sperm (35) outside of the heterogeneous inseminate (26) as compared to increased level of fertility (45) inside of the heterogeneous inseminate (26) may be due to a number of factors.

For example, there may be a lesser concentration of sperm outside of the heterogeneous inseminate (26), or the first amount of sperm (34) having greater initial level of fertility (44) may contribute, supplement or afford within the heterogeneous inseminate (26) a physical stimulus or chemical stimulus that acts upon the second amount of sperm (35) to generate the increased level of fertility (45) within the heterogeneous inseminate (26), or the second amount of sperm (35) may exhibit an increased level of motility (46) within the heterogeneous inseminate (26) as compared to outside of the heterogeneous inseminate (26), such greater motility can act to increase fertility of the second amount of sperm (35) in the heterogeneous inseminate (26).

Now referring primarily to FIG. 8 and FIG. 9, embodiments of the heterogeneous inseminate (3) can further include a first amount of sperm (34) and a second amount of sperm (35) (or a plurality of amounts of sperm (1)) from a corresponding first male animal (36) and a second male animal (37) (or plurality of male animals (2)) of the same species in which the first amount of sperm (35) or the second amount of sperm (36) or both the first amount of sperm (35) and the second amount of sperm (36) (or plurality of amounts of sperm (1)) can be sex-selected sperm (24).

Now referring primarily to FIG. 8, as to particular embodiments of the invention, the sex-selection process (47) above described and shown in FIGS. 1 and 2 for production of sex-selected sperm (24) can occur after combining the first amount of sperm (34) with the second amount of sperm (35) (or after combining a plurality of amounts of sperm (1)) to achieve the heterogeneous inseminate (26). After combination, the heterogeneous inseminate (26) can then undergo the sex selection process (47) resulting in a sex-selected heterogeneous inseminate (39) containing X-chromosome bearing sperm (18) and a sex-selected heterogeneous inseminate (39) containing Y-chromosome bearing sperm (19). The sex-selected heterogeneous inseminate (39) can contain X-chromosome bearing sperm (18) of the first amount of sperm (35) and the second amount of sperm (36) (or the plurality of amounts of sperm (1)) in similar proportion or concentration as in the original heterogeneous inseminate (26) to afford the substantial advantages above-described.

Similarly, the sex-selected heterogeneous inseminate (39) can contain Y-chromosome bearing sperm (19) of the first amount of sperm (34) and the second amount of sperm (35) (or plurality of amounts of sperm (1)) in similar proportion or concentration to the original heterogeneous inseminate (26) to afford the substantial advantages above-described. The sex-selected heterogeneous inseminate (39) containing X-chromosome bearing sperm (18) or the sex-selected heterogeneous inseminate (29) containing Y-chromosome bearing sperm (19) can be used in-vitro or in-vivo to fertilize the eggs (27) of a female animal (29) of the same species to produce sex-selected in-vivo embryos (48) or sex-selected offspring (30) or produce sex-selected in-vitro embryos (28) of a pre-selected sex. While FIGS. 8 and 9, show a sex-selected heterogeneous inseminate (39) containing only X-chromosome bearing sperm (18) of two or more male animals (2) being used for in-vitro (42) and in-vivo artificial insemination (31) of eggs (27); the invention is not so limited, and as above described embodiments of the sex-selected homogeneous inseminate (39) can include the X-chromosome bearing sperm (18) of two or more male animals (2) or the Y-chromosome bearing sperm (19) of two or more male animals (2).

Now referring primarily to FIG. 9, as to particular embodiments of the invention, the sex-selection process (47) for production of sex-selected sperm cells (24) can occur prior to combining the first amount of sperm (34) with the second amount of sperm (35) (or prior to combining the plurality of amounts of sperm (1)) to produce a sex-selected heterogeneous inseminate (39). As to these embodiments, each of the first amount of sperm (34) and the second amount of sperm (35) (or the plurality of amounts of sperm (1)) separately undergo the sex selection process (47) prior to combination. The first amount of sperm (34) can be separated into a subpopulation of X-chromosome bearing sperm (18) and into a subpopulation of Y-chromosome bearing sperm (19) as above described. Similarly, the second amount of sperm (35) (or each of the plurality of amounts of sperm (1)) can be separated into isolated subpopulations respectively containing X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19). An amount of the X-chromosome bearing sperm (18) obtained from the first amount of sperm (34) can be combined with an amount of the X-chromosome bearing sperm (18) obtained from the second amount of sperm (35) (or the X-chromosome bearing sperm (18) of a plurality of amounts of sperm (1) can be combined) to provide the sex-selected heterogeneous inseminate (39) containing X-chromosome bearing sperm (18).

Similarly, an amount of Y-chromosome bearing sperm (19) obtained from the first amount of sperm (34) can be combined with an amount of the Y-chromosome bearing sperm (19) obtained from the second amount of sperm (35) (or the Y-chromosome bearing sperm (18) of a plurality of amounts of sperm (1) can be combined) to provide a sex-selected heterogeneous inseminate (39) containing Y-chromosome bearing sperm (19).

The amount of the X-chromosome bearing sperm (18) obtained from the first amount of sperm (34) can be adjusted in relation to the amount of the X-chromosome bearing sperm (18) obtained from the second amount of sperm (35) (or the X-chromosome bearing sperm (18) obtained from each of the plurality of amounts of sperm (1) can be adjusted in relation to each other) to achieve a desired concentration or proportion of X-chromosome bearing sperm (18) from the first amount of sperm (34) and the second amount of sperm (35) (or the plurality of amounts of sperm (1)) in the sex-selected heterogeneous inseminate (39) to afford the substantial advantages above-described.

Similarly, the amount of the Y-chromosome bearing sperm (19) obtained from the first amount of sperm (34) can be adjusted in relation to the amount of the Y-chromosome bearing sperm (19) obtained from the second amount of sperm (35) (or the Y-chromosome bearing sperm (19) obtained from each of the plurality of amounts of sperm (1) can be adjusted in relation to each other) to achieve a desired concentration or proportion of Y-chromosome bearing sperm (19) from the first amount of sperm (34) and the second amount of sperm (35) (or the plurality of amounts of sperm (1)) in the sex-selected heterogeneous inseminate (26) to afford the substantial advantages above-described.

The sex-selected heterogeneous inseminate (39) containing X-chromosome bearing sperm (18) or the sex-selected heterogeneous inseminate (39) containing Y-chromosome bearing sperm (19) (or a particular proportion of X-chromosome bearing sperm (18) to Y-chromosome bearing sperm (19)) can be used for in-vitro fertilization (42) or in-vivo artificial insemination (31) to fertilize the eggs (27) of a female animal (29) of the same species to produce in-vivo sex-selected embryos (49) or in-vitro sex-selected embryos (50) of a pre-selected sex.

In regard to the increased level of fertility (45), particular embodiments of the invention can include a first amount of sperm (34) which outside of the heterogeneous inseminate (26) can have an initial level of fertility (44) of zero or nearly zero for a particular application, which can be due to insufficient numbers of sperm, insufficient motility of sperm, or insufficient physical or chemical stimuli which can be afforded within the homogeneous inseminate (26). Accordingly, once the threshold as to numbers of sperm, motility of sperm or the physical or chemical stimuli is achieved within the heterogeneous inseminate (26), the increased level of fertility (45) of the second amount of sperm (35) can be sufficient to allow successful fertilization of an egg (27) of a female animal (29) for that particular application, which would otherwise be unsuccessful or have a lesser fertility rate.

Understandably, the initial level of fertility (44) can be any level of fertility within a particular application, and the increased level of fertility (45) can be a comparable increase of fertility for that particular application. One measure by which initial level of fertility (44) and increased level of fertility (45) can be assessed is by measure of the initial level of motility (51) of the second amount of sperm (35) (or of any of the plurality of amounts of sperm (1)) outside of the heterogeneous inseminate (26) and the increased level of motility (46) of the second amount of sperm (36) (or of any of the plurality of amounts of sperm (1)) within the heterogeneous inseminate (26)). As to embodiments of the inventive heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (393) the increase in the motility of the second amount of sperm (35) within the heterogeneous inseminate (26) (or sex-selected heterogeneous inseminate (39)) as compared to outside of the heterogeneous inseminate (26) can be in the range of between about 1% and about 30%. With respect to sex-selected heterogeneous inseminates (39) as one example, the initial level of motility (51) of sex-selected sperm (24) outside of the sex-selected heterogeneous inseminate (26) shortly after separation by a sex-selection process (47), such as flow cytometry, can be at or below 45% as compared to the increased level of motility (46) of the an substantially identical sample of sex-selected sperm (24) within the heterogeneous inseminate (26) of about 60% to about 70%.

For the purposes of this invention, the term “extender” refers to a solution that comes in contact with the plurality of sperm cells (1), the heterogeneous inseminate (26), or sex-selected sperm (24), or sex-selected heterogeneous inseminate (39), typically for the purpose of dilution or as a cryoprotectant. Typical examples of an extender (52) can contain one or more of: sodium citrate, Tris[hydroxymethyl]aminomethane (also referred to as “TRIS”), TES (N-Tris [Hydroxymethyl]methyl-2-aminoethanesulfonic acid), monosodium glutamate, HEPES medium such as HEPES buffered medium, HEPES buffered bovine gamete medium and particularly HBGM3 and can further contain cryoprotectants (55) such as glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol; other organic substances such as egg yolk, an egg yolk extract, milk, a milk extract, casein, albumin, lecithin, bovine serum albumin, cholesterol; sugars such as the monosacharides, glucose, fructose, or mannose; detergents such as sodium dodecyl sulfate; antioxidants such butylated hydroxytoluene; capacitation facilitators such as alpha amylase, beta amylase, or beta glucuronidase; antibiotics such as tylosin, gentamicin, lincomycin, spectinomycin, linco-spectin (a combination of lincomycin and spectinomycin), penicillin, streptomycin, and ticarcillin; flow cytometer sheath fluids; and specifically without limiting the forgoing, particular extenders (52) referred to below as TRIS-A (53) and TRIS-B (54); although the inventive heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39) or methods of using the heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39) are not limited by the working examples which use TRIS-A (53) or TRIS-B (54) as an extender (52) to dilute concentration of the plurality of sperm cells (1), the sex-selected sperm cells (24) or embodiments of the heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39) which can be diluted by use of TRIS-A (53) or the use TRIS-B (54) as a cryoprotectant (55). The use of TRIS-A (53) or the use of TRIS-B (54) is described in detail as an illustrative example of the numerous and varied extenders that can be used with embodiments of the invention as diluents and cryoprotectants (55).

For the purposes of this invention the term “TRIS-A” refers to an extender (52) having the formulation in Table 7.

TABLE 7 TRIS-A Extender. TRIS 200 mM  Citric Acid 65 mM Fructose 56 mM Egg Yolk* 20% by volume Hydrochloric Acid addition to pH 6.8 *Egg Yolk can be clarified by winterization process to remove certain particulates and fats.

The formulation of TRIS-A set out in Table 7 can be from application to application of the invention modified to increase viability or reduce damage to the plurality of sperm cells (1), the heterogeneous inseminates (26) or sex-selected heterogeneous inseminates (39) and the above formulation is provided as a non-limiting example of a numerous and wide variety of similar extenders (52) which are suitable for use in making or using the embodiments of the invention. See also, Yassen, A. M. and Foote, R. H., Freezability of Bovine Spermatozoa in Tris-Buffered Yolk Extenders Containing Different Levels of Tris, Sodium, Potassium and Calcium Ions, J. Dairy Science, Vol. 50, No. 6, 887-892 (1966), hereby incorporated by reference in the entirety herein. Extenders (52) in general and specifically the particular extender TRIS-A (53) can further include one or more antibiotics (56) as above described or consistent with animal health regulations of any particular jurisdiction. Also, the description of TRIS-A (53) is not intended to be limiting with respect to the wide variety of extenders (52) which can be utilized in making and using certain embodiments of the invention, as described above.

For the purposes of this invention the term “TRIS-B” refers to an extender (52) having the formulation in Table 8.

TABLE 8 TRIS-B Extender. TRIS 200 mM  Citric Acid 65 mM Fructose 56 mM Egg Yolk* 20% by volume Glycerol 12% by volume Hydrochloric Acid addition to pH 6.8 *Egg Yolk can be clarified by winterization process to remove certain particulates and fats.

The formulation of TRIS-B (54) set out in Table 8 can from application to application of the invention be modified to increase viability or reduce damage to the plurality of sperm cells (1), the heterogeneous inseminates (26) or sex-selected heterogeneous inseminates (39) and the above formulation is provided as a non-limiting example of a numerous and wide variety of similar extenders (52) which are suitable for use in making or using the embodiments of the invention. Again see for example, Yassen, A. M. and Foote, R. H., Freezability of Bovine Spermatozoa in Tris-Buffered Yolk Extenders Containing Different Levels of Tris, Sodium, Potassium and Calcium Ions, J. Dairy Science, Vol. 50, No. 6, 887-892 (1966). Extenders (52) in general and specifically the particular extender TRIS-B (54) can further include one or more antibiotics (56) as above described or consistent with animal health regulations of any particular jurisdiction. Additionally, while TRIS-B (54) of the particular formulation set out in Table 8 uses glycerol as a cryoprotectant (55) the invention is not so limited. Also, the description of TRIS-B (54) is not intended to limiting with respect to the wide variety of extenders (52) which can be utilized in making and using certain embodiments of the invention, as described above.

Again referring primarily to FIGS. 8 and 9, as to particular embodiments, the plurality of sperm cells (1), the first amount of sperm (34) and the second amount of sperm (35) (whether or not sex-selected sperm (24)), the resulting heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39) can further include an amount of seminal fluid (57), which may be may be cell free semen (58) prepared by removal of entrained sperm (1) which may be diluted with an extender (52). Removal of entrained sperm (1) from the seminal fluid (57) can be achieved by centrifugation of semen at about 500×g for about 15 min. The seminal fluid (57) decanted and centrifuged a second time at 500×g for about 15 min.

Again referring to FIGS. 8 and 9, embodiments of the homogeneous inseminate (26) or sex-selected homogeneous inseminate (39) can further include a particular dosage form. As one non-limiting example, the dosage form for artificial insemination (31) of cattle can be a one-quarter cubic centimeter (0.25 cc) artificial insemination straw (25) which contains the heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39). While the FIGS. 8 and 9 only show a sex-selected heterogeneous inseminate (39) contained in the artificial insemination straw (25); the invention is not so limited and dosage forms whether in the form of an artificial insemination straw (25) or otherwise can contain a heterogeneous inseminate (26), a sex-selected heterogeneous inseminate (39), or can contain a combination of both a heterogeneous inseminate (26) and sex-selected heterogeneous inseminate (29).

Again referring to FIGS. 8 and 9, dosage forms of the heterogeneous inseminate (26) and the sex-selected heterogeneous inseminate (39) can be produced by similar steps or procedures as conventional insemination or sex-selected insemination dosage forms; however, the use of the heterogeneous inseminate (26) or sex-selected heterogeneous inseminate (39) can by comparison provide artificial insemination dosages (40) or in-vitro fertilization dosages (41) which exhibit an increased level of motility (46) or have an increased level of fertility (45).

Now referring primarily to FIG. 8, as one non-limiting example, a bovine embodiment of a sex-selected heterogeneous inseminate (39) can be produced by obtaining a first amount of sperm (34) (whether fresh or cryopreserved) of a first male animal (36) and obtaining a second amount of sperm (35) (whether fresh or cryopreserved) of a second male animal (37) of the same species of bovine male animal (or by obtaining a plurality of amounts of sperm (1) from a plurality of male animals (2) of the same species). The first amount of sperm (34), the second amount of sperm (35), or the plurality of amounts of sperm (1) can each be separately suspended in an amount of TRIS-A (53) (or other extender (52)) and then centrifuged in range of about 500 rounds per minute (“rpm”) and about 5,000 rpm for a period of time in a range of between about one minute and about ten min. The supernatant can be decanted from each of the amounts of sperm (34)(35)(1). Each of the amounts of sperm (34)(35)(1) can be extended with TRIS-A (53) (or other extender (52)) to achieve a concentration which allows each the extended first amount of sperm (34), second amount of sperm (35), or plurality of amounts of sperm (1) to be combined to produce a heterogeneous inseminate (26) each of the first amount of sperm (34), the second amount of sperm (35, or each of a plurality of amounts of sperm (1) at a desired concentration within the heterogeneous inseminate (26).

As a non-limiting example, the concentration of the first amount of sperm (34) and the second amount of sperm (35) within the heterogeneous inseminate (26) can be about 75 million sperm per milliliter. The concentration of the first amount of sperm (34) in the heterogeneous inseminate (26) can be about 55 million sperm per milliliter and the concentration of the second amount of sperm (35) in the heterogeneous inseminate (26) can be about 20 million sperm per milliliter; however the invention is not so limited, and the concentration of the first amount of sperm (34) or the second amount of sperm (35) or a plurality of amounts of sperm (1) within the heterogeneous inseminate (26) can be at any predetermined concentration depending upon the application, which as to certain embodiments can provide about equal concentration of each of a plurality of amounts of sperm (1) from a corresponding plurality of animals (2) or can provide unequal concentration as to a plurality of amounts of sperm (1) from a corresponding plurality of animals (2).

The heterogeneous inseminate (3) can then be incubated in about 38 micromolar Hoechst 33342 at about 75 million sperm/ml in the extender (52) for 1 hour at about 34° C.; although other stains or staining conditions can be utilized as above described, to achieve staining of the DNA of the first amount of sperm (34), the second amount of sperm (35), or the plurality of amounts of sperm (1) within the heterogeneous inseminate (26) for separation into subpopulations containing X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19), as above described or otherwise. As an example, the stained sperm contained in the heterogeneous inseminate (26) can be interrogated by laser excitation at 351 and 364 nm at 150 mW and based on the difference in fluorescent emission be separated into subpopulations of X-chromosome bearing sperm (18) and Y-chromosome bearing sperm (19) using a MOFLO flow cytometer (4) (or other makes or models of flow cytometers or multiple flow channel apparatus), as above described. The subpopulations of X chromosome-bearing sperm (18) and Y-chromosome bearing sperm (19) of about 90% purity as verified by resort analysis can be collected at between about 1,500 live sperm/second and about 5,000 live sperm/second into 2-ml Eppendorf tubes containing 100 micromolar Cornell Universal Extender (CUE) with 20% egg yolk.

Collected sex-selected sperm (24) can be centrifuged at about 600×g for 10 min and resuspended in TRIS B (54) to a concentration of about 2 million motile sex-selected sperm/mL to provide an embodiment of a sex-selected heterogeneous inseminate (39). The sex-selected heterogeneous inseminate (39) can be cooled to about 5° C. over about 75 min and artificial insemination dosages (40) transferred into 0.25-cc artificial insemination straws (25) (about 150 microliters to about 200 microliters per straw). Straws (25) can be cryopreserved, as above described by conventional procedures, or transported at about 3° C. to about 5° C. for use in artificial insemination (31) of female animals (29). Artificial insemination dosages (40) and control dosages can be inseminated using side-opening blue sheaths (IMV), one half of each straw into each uterine horn of female animal (29) of the same bovine species (total of about 3.5×10⁵ sex-selected sperm per artificial insemination dosage or about 250,000 sex-selected sperm from the first amount of sperm (34) and about 50,000 sex-selected sperm from the second amount of sperm (35)). Understandably, the respective concentration of sex-selected sperm (24) in an artificial insemination dosage (40) between a first amount of sperm (34), a second amount of sperm (35), or a plurality of amounts of sperm (1) can be adjusted by adjusting the initial concentration of the a sex-selected heterogeneous inseminate (39) prior to division into the corresponding plurality of artificial insemination dosages (40) or other dosage forms.

Now referring primarily to FIG. 9, as a general non-limiting example, a bovine embodiment of a sex-selected heterogeneous inseminate (39) can be produced by obtaining a first amount of sperm (34) as sex-selected sperm cells (24) (whether fresh or cryopreserved) of a first male animal (36) of a bovine species. The first amount of sperm (34) can be suspended in an amount of TRIS-A (53) (or other extender (52)) and then centrifuged in range of about 500 rounds per minute (“rpm”) and about 5,000 rpm for a period in a range of between about one minute and about ten min. The supernatant can be decanted. The first amount of sperm (34) can be extended with TRIS-A (53) extender (or other extender (52)) to achieve a concentration of the extended first amount of sperm (34) of about four times greater than the expected concentration of the second amount of sperm (35), or other concentration depending upon the application. The four times concentration of the first amount of sperm (34) in TRIS-A (54) (or other extender (53)) can be for example a concentration in a range of about 3.2 million sperm cells/mL and about 128 million sperm cells/mL.

The extended first amount of sperm (34) (sex-selected sperm (24)) can be cooled to a temperature in a range of about 4 degrees Celsius (“° C.”) and about 5° C. The cooled extended first amount of sperm (34) can be held at this temperature to allow the membranes of the first amount of sperm (34) to move toward equilibrium or equilibrate with the TRIS-A (53) (typically a period of about 90 min or as to certain embodiments not less than 90 min). The cooled extended first amount of sperm (34) can be held in this condition not to exceed a length of time in which the sperm cells (34) remain viable or capable of fertilizing an egg (27) of a female animal (29) of the same species as the first and second male animals (36)(37). Typically, the period of time held will not exceed 12 h.

A second amount of sperm (35) as sex-selected sperm cells (24) (fresh or cryopreserved) can be obtained of a second male animal (37) of the same bovine species. The second amount of sperm (35) can be suspended in an amount of TRIS-A (53) (or other extender (52)) and then centrifuged in range of about 500 rounds per minute (“rpm”) and about 5,000 rpm for a period in a range of between about one minute and about ten min. The supernatant can be decanted and the pellet containing the second amount of sperm (35) can be suspended in an amount of TRIS-A (53) to achieve a concentration of the second amount of sperm (35) of about one-fourth that of the first amount of sperm (34). Accordingly, the second amount of sperm (35) as a non-limiting example can have a concentration in a range of about 800,000 sperm per milliliter and about 32 million sperm per milliliter depending upon various factors such as the species of the second male animal (37), the scarcity of the sperm, the fertility of the sperm, the dosage form, the method of insemination (whether artificial insemination (31) or in-vitro fertilization (42)), whether for multiple ovulation embryo transfer or single ovulation single embryo production, the female animal (29) of the same species as the first and second male animal (36)(37) being inseminated, or the like.

The extended second amount of sperm (35) can be cooled to a temperature in a range of about 4° C. and about 5° C. The cooled extended second amount of sperm (35) can be held at this temperature to allow the membranes of the second amount of sperm (35) to move toward equilibrium or equilibrate with the TRIS-A (53) (typically a period of about 90 min or as to certain embodiments not less than 90 min).

About equal volumes of the cooled extended first amount of sperm (34) (sex-selected sperm (24)) and the cooled extended second amount of sperm (35) (sex-selected sperm (24)) can be combined to achieve two times greater concentration of the first amount of sperm (34) with respect to the final concentration of the second amount of sperm (35) contained in the resulting heterogeneous sex-selected inseminate (39).

To the resulting sex-selected heterogeneous inseminate (39) about an equal volume of TRIS-B (54) (containing between about 12% and 20% glycerol) can be added and the combination cooled to temperature in a range of about 4° C. and about 5° C. The cooled extended sex-selected heterogeneous inseminate (39) can be held at this temperature to allow the membranes of the sperm cells (1)(24) to move toward equilibrium or equilibrate with the TRIS-B (54) (typically a period in the range of about 30 min and about 90 min or as to certain embodiments not less than 90 min). The cooled sex-selected heterogeneous inseminate (39) can be held in this condition not to exceed a length of time in which the sex-selected sperm cells (24) remain viable or capable fertilizing an egg (27) of a female (29) of the same species as the male animals (2).

The cooled sex-selected heterogeneous inseminate (39) can be handled by normal procedures utilized for producing artificial insemination doses (40). Accordingly, as to certain embodiments, the cooled sex-selected heterogeneous inseminate (39) can be aliquoted into 0.25 cc artificial insemination straws (25) (about 100 microliters to about 200 microliters per straw). The 0.25 cc artificial insemination straw (25) can contain an amount of the sex-selected heterogeneous inseminate (39) which contains sex-selected sperm (24) from the second amount of sperm (35) in the range of about 50,000 and about 2 million and from the first amount of sperm (14) in the range of about 200,000 and about 8 million. However, a greater or lesser number of the first amount of sperm (34) and the second amount of sperm (35) can be included in each 0.25 cc artificial insemination straw (25) by adjusting the initial concentrations of the first amount of sperm (34) and the second amount of sperm (35) with the total number of sex-selected sperm (24) in each artificial insemination dosage (40) of the instant example being in the range of about 250,000 and about 10 million sex-selected sperm (24).

The sex-selected heterogeneous inseminate (39) can used for artificial insemination (31) to fertilize the egg(s) (27) of a female animal (29) of the same species as the first and second male (36)(37) for the production of sex-selected in-vivo embryo (49) whether as single embryo pregnancies to generate offspring (30) for meat or animal replacement or multiple embryo pregnancies for MOET by subsequent flushing of a plurality of sex-selected embryos (49). Alternately, the sex-selected heterogeneous inseminate (39) can be used in-vitro fertilization procedures (42) to fertilize eggs (27) obtained from a female animal (297) of the same species to produce in vitro sex-selected in-vitro fertilized embryos (50).

The use of heterogeneous sex-selected inseminate (39) can result in increased fertility rates. As one non-limiting example, where a plurality of amounts of sperm (1) are processed in accordance with either the method shown in FIG. 8 or FIG. 9 and as described above to produce a sex-selected heterogeneous inseminate (39), the sex-selected heterogeneous inseminate (39) can have an increased level of fertility (45) overall as compared to any initial level of fertility (44) for any one of the plurality of amounts of sperm (1) outside of the sex-selected heterogeneous inseminate (39).

As a second non-limiting example, where a plurality of amounts of sperm (1) from a corresponding plurality animals (2) of the same species are processed as above described to provide a sex-selected heterogeneous inseminate (39) which includes at least one amount of sex-selected sperm (24) having a comparably greater initial level of fertility (45) to one or more amounts of sex-selected sperm (24) having a lesser initial level of fertility (44) outside of the sex-selected heterogeneous inseminate (26), the one or more of the amounts of sex-selected sperm (24) within the sex-selected heterogeneous inseminate (39) can exhibit an increased level of fertility (45) which can exceed the initial level of fertility (44) of the amount of sex-selected sperm (24) having the greater initial level of fertility (45).

As a third non-limiting example, the seminal fluid (57) of one or more amounts of sex-selected sperm (24) which exhibit a comparably greater initial level of fertility (45) outside of the sex-selected heterogeneous inseminate (26) can be stored as an amount cell free semen (58) and added back into the sex-selected heterogeneous inseminate (39) processed in accordance with either the method shown in FIG. 8 or FIG. 9 and as described above to produce a sex-selected heterogeneous inseminate (39). The amount of cell free semen (58) in a sex-selected heterogeneous inseminate (39) can be in the range of zero cell free semen to about 2 parts cell free semen (58) to one part sex-selected heterogeneous insemination (39). The concentration of the sex-selected sperm (24) can be adjusted as above-described to afford the desired concentration of sex-selected sperm (24) in an artificial insemination dosage (40) or an in-vivo insemination dosage (40).

The invention can further include a mammal produced in accordance with any of the above described embodiments of the invention, or can include a mammal of predetermined sex in accordance with the various embodiments of the invention that provide sperm cell insemination samples having an enriched population of either X-chromosome bearing sperm cells or enriched population of Y-chromosome bearing sperm cells, or a mammal produced in accordance with any embodiment of the invention in which a sperm cell insemination sample containing a low number of sperm cells compared to the typical number used to inseminate that particular species of mammal is used, or elk progeny produced in accordance with the invention as described above.

As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both a sperm cell process system including both techniques as well as devices to accomplish sperm cell processing. In this application, various sperm cell processing techniques are disclosed as part of the results shown to be achieved by the various devices described and as steps which are inherent to utilization. They are simply the natural result of utilizing the devices as intended and described. In addition, while some devices are disclosed, it should be understood that these not only accomplish certain methods but also can be varied in a number of ways. Importantly, as to all of the foregoing, all of these facets should be understood to be encompassed by this disclosure.

The discussion included in this nonprovisional application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terminology, each element of the device implicitly performs a function. Apparatus claims may not only be included for the device described, but also method or process claims may be included to address the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of the claims which will be included in a full patent application.

It should also be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fall within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) shown, the great variety of implicit alternative embodiments, and the broad methods or processes and the like are encompassed by this disclosure.

Further, each of the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may be expressed by equivalent apparatus terms or method terms—even if only the function or result is the same. Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of a “flow-sorter” should be understood to encompass disclosure of the act of “flow-sorting”—whether explicitly discussed or not—and, conversely, were there effectively disclosure of the act of “flow-sorting”, such a disclosure should be understood to encompass disclosure of a “flow-sorter” and even a “means for flow-sorting” Such changes and alternative terms are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentioned in this application for patent; or patents, publications, or other references mentioned in this application for patent are hereby incorporated by reference. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. Finally, all references listed in the list of References To Be Incorporated By Reference In Accordance With The Provisional Patent Application or other information statement filed with the application are hereby appended and hereby incorporated by reference, however, as to each of the above, to the extent that such information or statements incorporated by reference might be considered inconsistent with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicant(s).

Thus, the applicant(s) should be understood to claim at least: i) each of the sperm cell processing devices as herein disclosed and described, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods, iv) those alternative designs which accomplish each of the functions shown as are disclosed and described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, and ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the elements disclosed, and xi) each potentially dependent claim or concept as a dependency on each and every one of the independent claims or concepts presented. In this regard it should be understood that for practical reasons and so as to avoid adding potentially hundreds of claims, the applicant may eventually present claims with initial dependencies only. Support should be understood to exist to the degree required under new matter laws—including but not limited to European Patent Convention Article 123(2) and United States Patent Law 35 USC 132 or other such laws—to permit the addition of any of the various dependencies or other elements presented under one independent claim or concept as dependencies or elements under any other independent claim or concept Further, if or when used, the use of the transitional phrase “comprising” is used to maintain the “open-end” claims herein, according to traditional claim interpretation. Thus, unless the context requires otherwise, it should be understood that the term “comprise” or variations such as “comprises” or “comprising”, are intended to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Such terms should be interpreted in their most expansive form so as to afford the applicant the broadest coverage legally permissible.

For the purposes of this invention, the term “animal” means a species of mammal, ayes, or fish from which semen containing an amount of sperm is produced by a male animal of the species and an egg is produced by the female animal of the species and a mammal, without limiting the general forgoing statement, can be a species of: horse, cattle, camel, deer, pig, sheep, goat, dog, cat, lion, whale, dolphin, porpoise, seal, hare, rabbit, elephant, mouse, rat, or the like, and a fish, without limiting the general forgoing statement, can be a species of: salmon, tuna, sturgeon, halibut, catfish, or the like, and a bird, without limiting the general forgoing statement can be a species of: chicken, turkey, eagle, falcon, ostrich, emu, duck, goose, or the like.

For the purposes of this invention the term “semen” means seminal fluid which may contain sperm (also referred to as “spermatozoa”) secreted by the gonads of a male animal which can be collected from the male animal by a variety of methods such as use of an artificial vagina, manual manipulation of the penis, electrical manipulation of the anus, or the like.

For the purposes of this invention the term “sperm” or “sperm cell” means the haploid cell that is the garnet of a male animal which may join an egg (also referred to as “ovum”) to produce a zygote and broadly encompasses infertile sperm, sperm having a comparably lesser or a comparably greater fertility between a first amount of sperm obtained from a first animal and a second amount of sperm obtained from a second animal and which may be obtained in the form of a raw ejaculated semen, frozen semen, as sperm separated from the semen and contained in an extender or diluent, or as sex-selected sperm.

For the purposes of this invention the term “sex-selected sperm” means sperm which have been separated, regardless as to the method of separation, into subpopulations containing X-chromosome bearing sperm and Y-chromosome bearing sperm having a purity in the range of about 70 percent (“%”) and about 100%.

For the purposes of this invention, the term “inseminate” or “insemination sample” is intended to broadly encompass an amount of sperm whether contained in semen or other extender or diluent which can be utilized to fertilize the eggs of a female animal whether in-vitro or in-vivo and without limitation to the general forgoing statement can be an amount of sperm whether or not sex-selected introduced or injected into the reproductive tract of a female animal whether by artificial insemination procedures or otherwise, or utilized in in-vitro fertilization procedures.

For the purposes of this invention, the term “heterogeneous inseminate” means an inseminate in the form of a combination of a first amount sperm obtained from a first male animal and a second amount of sperm obtained from a second male animal, the first animal and the second male animal being of the same species; however, the invention is not so limited and a plurality of amounts of sperm obtained from a corresponding plurality of male animals of the same species can be combined to produce various embodiments of the heterogeneous inseminate.

For the purposes of this invention, the term “sex-selected heterogeneous inseminate” means a heterogeneous inseminate having the first amount of sperm obtained from a first male animal and the second amount of sperm obtained from a second male animal (or a plurality of amounts of sperm obtained from a plurality of animals) which are processed, whether prior to combination or after combination, such that the first amount of sperm and the second amount of sperm (or the plurality of amounts of sperm) contained in a heterogeneous inseminate are sex-selected sperm.

For the purposes of this invention, the term “fertility” means the fertilization rate determined for an artificial insemination procedure as the number of pregnancies achieved within a population of inseminated female animals divided by the total number inseminated female animals and multiplied by 100 or determined for in-vitro fertilization procedure as the number of embryos (greater than or equal to two cells) divided by the sum of the embryos and oocytes recovered and multiplied by 100.

For the purposes of this invention, the term “motility” means moving sperm in an inseminate or sample of sperm.

For the purposes of this invention, the term “infertile” means as to any particular procedure incapable of fertilizing an egg of a female animal. 

We claim:
 1. A method of producing a heterogeneous inseminate, comprising: obtaining a first amount of sperm of a first animal; obtaining a second amount of sperm of a second animal, said first animal and said second animal of the same species, wherein said first amount of sperm of said first animal or said second amount of sperm of said second animal comprises sex-selected sperm; combining said first amount of sperm of said first animal with said second amount of sperm of said second animal; and adjusting said first amount of sperm of said first animal in said heterogeneous inseminate to provide a concentration sufficient to increase motility of said second amount of sperm of said second animal in said heterogeneous inseminate.
 2. The method of producing a heterogeneous inseminate of claim 1, wherein said first amount of sperm of said first animal has a greater fertility than said second amount of sperm.
 3. The method of producing a heterogeneous inseminate of claim 1, wherein a concentration of said first amount of sperm of said first animal in said heterogeneous inseminate has a greater concentration than said second amount of sperm of said second animal.
 4. The method of producing a heterogeneous inseminate of claim 1, wherein said first amount of sperm of said first animal comprises an amount of infertile sperm.
 5. The method of producing a heterogeneous inseminate of claim 1, wherein said first amount of sperm and said second amount of sperm each comprise said sex-selected sperm.
 6. The method of producing a heterogeneous inseminate of claim 5, further comprising selecting sex of said first amount of sperm of said first animal and selecting sex of said second amount of sperm of said second animal prior to said step of combining said first amount of sperm of said first animal with said second amount of sperm of said second animal.
 7. The method of producing a heterogeneous inseminate of claim 5, further comprising selecting sex of said first amount of sperm of said first animal and said second amount of sperm of said second animal after said step of combining said first amount of sperm of said first animal with said second amount of sperm of said second animal.
 8. The method of producing a heterogeneous inseminate of claim 5, wherein said first amount of sperm of said first animal or said second amount of sperm of said second animal comprises frozen-thawed sperm.
 9. The method of producing a heterogeneous inseminate of claim 5, wherein said species of animal is selected from the group consisting of: a mammal, an aves, and a fish.
 10. The method of producing a heterogeneous inseminate of claim 5, wherein said species of animal is a bovine mammal and wherein said second amount of sperm of said second animal in said heterogeneous inseminate has a concentration of between about 2×10⁴ sperm per milliliter and about 4×10⁶ sperm per milliliter and said first amount of sperm of said first animal in said heterogeneous inseminate has a concentration of between 2×10⁷ sperm per milliliter and about 8×10⁷ sperm per milliliter.
 11. The method of producing a heterogeneous inseminate of claim 10, further comprising producing said heterogeneous inseminate in a dosage form having a volume between one-tenth milliliter and about one milliliter contained in an artificial insemination straw.
 12. The method of producing a heterogeneous inseminate of claim 11, further comprising freezing said dosage form having a volume of between about one-tenth milliliter and about one milliliter contained in said artificial insemination straw.
 13. The method of producing a heterogeneous inseminate of claim 1, wherein said adjusting of said first amount of sperm of said first animal in said heterogeneous inseminate increases fertility of said second amount of sperm of said second animal, and wherein fertility is determined as the ability to fertilize an egg of said species of animal.
 14. The method of producing a heterogeneous inseminate of claim 1, further comprising admixing an amount of cell free seminal fluid prepared from an amount of semen having a greater initial level of fertility than said level of fertility of said first amount of sperm and/or said second amount of sperm.
 15. A method of producing a heterogeneous inseminate, comprising the steps of: obtaining a first amount of sperm of a first animal; obtaining a second amount of sperm of a second animal, said first animal and said second animal of the same species, wherein said first amount of sperm of said first animal or said second amount of sperm of said second animal comprises sex-selected sperm; combining said first amount of sperm of said first animal with said second amount of sperm of said second animal; and adjusting said first amount of sperm of said first animal in said heterogeneous inseminate to provide a concentration sufficient to increase fertility of said second amount of sperm of said second animal in said heterogeneous inseminate.
 16. The method of producing a heterogeneous inseminate of claim 15, wherein said first amount of sperm of said first animal has a greater fertility than said second amount of sperm.
 17. The method of producing a heterogeneous inseminate of claim 15, wherein a concentration of said first amount of sperm of said first animal in said heterogeneous inseminate has a greater concentration than said second amount of sperm of said second animal.
 18. The method of producing a heterogeneous inseminate of claim 15, wherein said first amount of sperm of said first animal comprises an amount of infertile sperm.
 19. The method of producing a heterogeneous inseminate of claim 15, wherein said first amount of sperm and said second amount of sperm each comprise said sex-selected sperm.
 20. The method of producing a heterogeneous inseminate of claim 19, further comprising selecting sex of said first amount of sperm of said first animal and selecting sex of said second amount of sperm of said second animal prior to said step of combining said first amount of sperm of said first animal with said second amount of sperm of said second animal.
 21. The method of producing a heterogeneous inseminate of claim 19, further comprising selecting sex of said first amount of sperm of said first animal and said second amount of sperm of said second animal after said step of combining said first amount of sperm of said first animal with said second amount of sperm of said second animal.
 22. The method of producing a heterogeneous inseminate of claim 19, wherein said first amount of sperm of said first animal and/or said second amount of sperm of said second animal comprises frozen-thawed sperm.
 23. The method of producing a heterogeneous inseminate of claim 15, wherein said species of animal is selected from the group consisting of: a mammal, an ayes, and a fish.
 24. The method of producing a heterogeneous inseminate of claim 15, wherein said species of animal is a bovine mammal and wherein said second amount of sperm of said second animal in said heterogeneous inseminate has a concentration of between about 2×10⁴ sperm per milliliter and about 4×10⁶ sperm per milliliter and said first amount of sperm of said first animal in said heterogeneous inseminate has a concentration of between 2×10⁷ sperm per milliliter and about 8×10⁷ sperm per milliliter, and wherein said second amount of sperm comprises sex-selected sperm.
 25. The method of producing a heterogeneous inseminate of claim 24, wherein said mammal comprises a bovine mammal and further comprising the step of producing said heterogeneous inseminate in a dosage form having a volume of between about one-tenth milliliter and about one milliliter contained in an artificial insemination straw.
 26. The method of producing a heterogeneous inseminate of claim 25, further comprising freezing said dosage form having a volume of between about one-tenth milliliter and about one milliliter contained in said artificial insemination straw.
 27. The method of claim 15, wherein fertility is determined as the ability to fertilize an egg of said species of animal.
 28. The method of producing a heterogeneous inseminate of claim 15, further comprising admixing an amount of cell free seminal fluid prepared from an amount of semen having a greater initial level of fertility than said level of fertility of said first amount of sperm and/or said second amount of sperm. 